1. Field of the Invention
The present invention relates to the field of optical analysis of fluorescent particles in fluid streams.
2. Description of Related Art
Flow-type particle analyzers, such as flow cytometers, are well known analytical tools that enable the characterization of particles on the basis of optical parameters such as light scatter and fluorescence. In a flow cytometer, for example, particles, such as molecules, analyte-bound beads, or individual cells, in a fluid suspension are passed by a detection region in which the particles are exposed to an excitation light, typically from one or more lasers, and the light scattering and fluorescence properties of the particles are measured. Particles or components thereof typically are labeled with fluorescent dyes to facilitate detection, and a multiplicity of different particles or components may be simultaneously detected by using spectrally distinct fluorescent dyes to label the different particles or components. Typically, a multiplicity of photodetectors, one for each of the scatter parameters to be measured, and one for each of the distinct dyes to be detected. The data obtained comprise the signals measured for each of the light scatter parameters and the fluorescence emissions.
Cytometers further comprise means for recording the measured data and analyzing the data. For example, typically, data storage and analysis is carried out using a computer connected to the detection electronics. The data typically are stored in tabular form, wherein each row corresponds to data for one particle, and the columns correspond to each of the measured parameters. The use of standard file formats, such as an “FCS” file format, for storing data from a flow cytometer facilitates analyzing data using separate programs and machines. Using current analysis methods, the data typically are displayed in 2-dimensional (2D) plots for ease of visualization, but other methods may be used to visualize multidimensional data.
The parameters measured using a flow cytometer typically include the excitation light that is scattered by the particle along a mostly forward direction, referred to as forward scatter (FSC), the excitation light that is scattered by the particle in a mostly sideways direction, referred to as side scatter (SSC), and the light emitted from fluorescent molecules in one or more channels (range of frequencies) of the spectrum, referred to as FL1, FL2, etc., or by the fluorescent dye that is primarily detected in that channel Different cell types can be identified by the scatter parameters and the fluorescence emissions resulting from labeling various cell proteins with dye-labeled antibodies.
Flow cytometers are commercially available from, for example, BD Biosciences (San Jose, Calif.). Flow cytometry is described at length in the extensive literature in this field, including, for example, Landy et al. (eds.), Clinical Flow Cytometry, Annals of the New York Academy of Sciences Volume 677 (1993); Bauer et al. (eds), Clinical Flow Cytometry: Principles and Applications, Williams & Wilkins (1993); Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); and Shapiro, Practical Flow Cytometry, 4th ed., Wiley-Liss (2003); all incorporated herein by reference. The data obtained from an analysis of cells (or other particles) by multi-color flow cytometry are multidimensional, wherein each cell corresponds to a point in a multidimensional space defined by the parameters measured. Populations of cells or particles are identified as clusters of points in the data space. The identification of clusters and, thereby, populations can be carried out manually by drawing a gate around a population displayed in one or more 2-dimensional plots, referred to as “scatter plots” or “dot plots, of the data. Alternatively, clusters can be identified, and gates that define the limits of the populations, can be determined automatically. A number of methods for automated gating have been described in the literature. See, for example, U.S. Pat. Nos. 4,845,653; 5,627,040; 5,739,000; 5,795,727; 5,962,238; 6,014,904; 6,944,338, each incorporated herein by reference.
In a typical laser-based flow cytometer, the excitation wavelengths available are limited by the availability of a suitable laser. Wavelength-selectable, single-wavelength excitation sources have been described for use in flow cytometry. For example, U.S. Pat. No. 4,609,286 (Sage) describes a flow cytometer that uses a dispersion prism to select a wavelength from a spectrally rich light source for use as the excitation source. The light source is dispersed by the prism such that the wavelength can be selected using a slit to allow only light of essentially a single wavelength through, block all other wavelengths. The desired wavelength can be selected by physically moving the slit to correspond to the desired wavelength in the spectrum.
Telford et al, 2009, Cytometry A 75(5):450-459, describes the use of a supercontinuum white light laser as an excitation source in flow cytometry. The supercontinuum white light laser emits continuously over a wide bandwidth ranging from the near-ultraviolet to the infrared, thus appearing white to the human eye. Telford et al. describe interposing an acoustooptical filter or a coated bandpass filter in front of the beam to isolate particular wavelength ranges, permitting the user to select bandwidths of interest from the supercontinuum. The resulting excitation source can be used to select any single excitation wavelength and bandwidth by using a filter with the desired color transmission requirements.
In a typical flow cytometer, fluorescence emissions are measured in a multiplicity of detection channels (each defined as a range of frequencies within the spectrum), wherein the emissions in each channel are measured using a single photodetector. Thus, each detector provides a single measure of a range of frequencies. Typically, the detector channels are selected such that each channel is optimized to detect emissions from one of the distinct dyes. Alternatively, the emission light can be measured using an array of detection channels such that each dye emissions are measured in more than one channel.
Robinson et al., in Advanced Biomedical and Clinical Diagnostic Systems III, edited by Tuan Vo-Dinh et al., Proc. of SPIE Vol. 5692 (SPIE, Bellingham, Wash., 2005): 3579-365, describes a flow cytometer detection system in which the emitted light is dispersed by diffraction grating onto a 32 channel PMT detector. Thus, fluorescence emissions are measured in 32 narrow, adjacent detection channels that together span a region of the spectrum. Instead of a single fluorescence intensity value for each dye, the data obtained using this system comprise, for each dye, intensity values for a multiplicity of adjacent detection channels. The set of measurements obtained from a dye across a multiplicity of spectrally adjacent detection channels depends on the emission spectrum of a dye.